The present invention concerns a new process for performing liquid chromatography in which there is a sequence of steps of which at least two consecutive steps (step 1 and step 2) are in fluidised bed mode by the use of an upward flow.
With respect to various modes of the invention reference is made to copending International Patent Application derived from SE 9803813-6 and SE 9803737-7. This International Patent Application is hereby incorporated by reference.
Liquid chromatographic processes are carried out on particle matrices in form of packed or fluidised beds. The processes typically contain at least one step according to type (b) below and one or more functional steps selected from the remaining types of steps (a,c,d,e,f):
a) equilibrating the particles with a liquid conditioning the particles for capture/binding;
b) capturing one or more compounds present in a liquid sample by the particles;
c) washing the particles to which said one or more compounds have become bound;
d) releasing at least one of said one or more compounds from the particles;
e) cleaning the particles; and
f) regenerating the particles.
The capture step (type b) together with the selected steps define an actual sequence in a particular chromatographic process. In an actual sequence there may also be steps other than those outlined above (a-f). A typical sequence comprise the sequence
a,b,c,d,e,f(a),b,c,d,e,f(a) . . . possibly with extra steps inserted in the sequence given. f(a) means that step a and step f may coincide and that chromatographic processes can be cyclic.
In each step the particles are treated with an appropriate liquid (solution/buffer) that is aqueous or non-aqueous.
The term xe2x80x9ccapturexe2x80x9d includes that the compound becomes bound to the particles. The binding may occur via the formation of affinity bonds, covalent bonds, entrapment within the particles etc. Examples of affinity are bioaffinity, ionic interaction, hydrophobic interaction etc. The captured compound may be a compound that is to be purified or a contaminant that one wants to separate from another compound or remove from the liquid used in the capture step.
The liquid used in the releasing step typically contains an agent that will release the captured compound, for instance a buffer giving an appropriate pH, a salt giving an appropriate ionic strength, a substance that competitively will inhibit the binding between the captured compound and an affinity ligand/structure on the particles, etc. The term xe2x80x9creleasexe2x80x9d includes release through breaking of affinity bonds, covalent bonds etc. Covalent bonds can be broken by chemical reactions or enzymatically.
The liquid used in a step can change continuously or step wise during a step. Releasing by the aid of a gradient, for instance, is typical for elution on packed beds but has been rare on fluidised beds (Shiloach et al., Sep. Sci. Techn. 34(1) (1999) 29-40). Another example is changing a washing solution during a washing step.
In packed beds, the releasing step typically can consist of one or more substeps. For instance the capture step may mean capture of two or more compounds that bind differently to the particles. For release, the compounds may require different conditions and different compositions of the liquid.
Steps can wholly or partly coincide. The regeneration step, for instance, is primarily related to regeneration of the particles to be used in a second cycle of the process and then coincides with the equilibration step of the second run. The capture step can mean that the compound is only retarded suggesting that the releasing step is at the same time ongoing. In case a contaminant is captured by the particles, possibly in combination with passing through the compound to be purified, release can take place in the cleaning step.
Cleaning steps are often called cip (=cleaning in place). Cip-steps normally comprise high concentration of solutes, such as NaOH, in the liquid used. This means that the liquid for cleaning often has the highest density in an actual sequence.
Each step can be run in a fluidised or packed bed mode with vertical flow that either may be upward or downward. The flow direction may switch between different steps. Plug flow has often been of advantage in chromatography, in particular in capture steps.
The same or different vessels can be used for the various steps of an actual sequence.
During the various steps the particles are placed in a vessel as known in the field. Se WO 9520427 (Amersham Pharmacia Biotech AB), WO 9218237 (Amersham Pharmacia Biotech AB), our copending International Patent Application derived from SE 9803813-6 and SE 9803737-7 etc. Suitable vessels have an inlet end and an outlet end. The vessel is typically placed vertically with the outlet pointing vertically upwards on the top side and the inlet pointing vertically downwards on the bottom side. It can also be the other way round. The inlet and outlet function, respectively, may comprise one or more openings into the vessel interior.
Density differences between liquids used in consecutive steps have been used previously in model experiments of fluidised bed purification. These experiments have included various concentrations of glycerol in the washing solution for small scale fluidised bed treatments. The purpose has been to increase the viscosity, and possibly also the density, of the washing solution compared to the solution applied for the adsorption/capture step. See Draeger and Chase, Bioseparation 2 (1991) 67-80; Chase et al, Sep. Sci. Techn. 27 (1992) 2021-2039; Chase et al, J. Chromatog. 597 (1992) 129-145; Chase et al, 6th European Congress of Biotechnology (ECB 6), Florence, Italy, Jun. 13-17, 1993; Chase, TIBTECH 12 (1994) 296-303; Chang et al, Biotechn. Bioengin. 48 (1995) 355-366; and Chang et al, Biotechn. Bioengin. 49 (1996) 204-216). The articles discuss that there are certain disadvantages on the subsequent elution (releasing) step due to the viscosity created by the added glycerol and that these disadvantages can be avoided by running the subsequent releasing step in a packed bed mode.
In fluidised bed chromatography liquids of increased densities have often been used when going from an equilibration step to a capture step (the samples are often is relatively dense).
Recently gradient elution has been applied in fluidised bed chromatography. See Shiloach et al., Sep. Sci. Techn. 34(1) (1999) 29-40.
In the releasing step of an actual sequence defined above the liquid used contains an agent that will release a captured compound from the particles. This means that the density of a liquid will tend to increase during the releasing step. In case the bed is fluidised by an upward flow there will be a tendency that liquid containing the released compound will be transported downwards simultaneously with the front of the release liquid progressing upwards. The result will be a dilution of the released compound and many times an unfavourable increase in the volume of liquid to be handled in the subsequent processing of the released compound.
Washing liquids may be relatively light. If a washing liquid has a density lower than the density of the liquid of a preceding step it will cause turbulence and lowered efficiency of the washing step. This is particularly pronounced for washing steps that are consecutive to a-capture step because the liquid used in a capture step many times is relatively dense.
These drawbacks will be more pronounced in vessels that do not have a movable outlet adapter compared to vessels that have this type of adapter.